Ice nucleus-forming bacterium strain Xanthomonas campestris FERM BP-4191 and process for cultivation of the same

ABSTRACT

The present invention provides a new ice nucleus-forming bacterium strain, Xanthomonas campestris INXC-1 (FERM BP-4191), a process for the cultivation of the new ice nucleus-forming bacterium, an ice nucleus-forming substance containing the ice nucleus-forming bacterium, and the uses of the ice nucleus-forming substance.

TECHNICAL FIELD

The present invention relates to a new strain of an ice nucleus-formingbacterium, a process for the cultivation of the new strain of the icenucleus-forming bacterium, an ice nucleus-forming substance containingthe strain, and the uses of the ice nucleus-forming substance.

BACKGROUND ART

As bacteria for promoting the formation of ice, Pseudomonas syringae andErwinia ananas have hitherto been well known. Also, among Xanthomonascampestris bacteria, those known are the following three strains: thestrain obtained from the leaves of Florida citrus [Goto Masao et al.,Nisshokubyo-Hou, 54, 196 (1988); and Minsavage G. V. & Stall R. E.,(1986), Proc. 6th Int. Conf. Plant Path. Bact., 994-1004], X. campestrispv. translucens [Kim H. K., Orser C., Lindow S. E. and Sands D. C.,Plant Disease, 71, 994-997 (1987)], and the strain obtained from teabuds (INAX) [Goto Masao et al., Nisshokubyo-Hou, 54, 189-197 (1988)].

However, among these ice nucleus-forming bacteria, Pseudomonas syringaewhich is utilized for practical use is known to have phytopathogenicity,so that it may adversely affect the environment when it is used forspraying to form artificial snow. On the other hand, bacteria having nosuch phytopathogenicity generally grow slowly and thus may not besuitable for production on an industrial scale. Additionally, bacteriawhich are negative with respect to tobacco hypersensitivity reaction[Phytopathology, 77, 611-615 (1987)] are generally free ofphytopathogenicity.

It would therefore be extremely advantageous to the industry to discovera strain which is negative with respect to tobacco hypersensitivityreaction and yet can grow at a high rate.

An object of the present invention is to provide a new icenucleus-forming bacterium which has the above advantages. Another objectof the present invention is to provide a process for the cultivation ofthe new ice nucleus-forming bacterium. Still another object of thepresent invention is to provide an ice nucleus-forming substancecontaining the new ice nucleus-forming bacterium and further to developuses thereof in various fields.

DISCLOSURE OF THE INVENTION

The present inventors have conducted extensive researches for thepurpose of attaining the above stated objects. As a result, they havefound that a bacterium strain obtained from tea leaves is a new strainwhich can fulfil the intended purpose, and have thus developed thepresent invention.

The present invention provides a new Xanthomonas campestris strain,INXC-1 (FERM BP-4191).

The present invention also provides a process for the cultivation ofXanthomonas campestris INXC-1.

The present invention further provides an ice nucleus-forming substancecontaining Xanthomonas campestris INXC-1 and presents uses thereof invarious fields.

BEST MODES FOR CARRYING OUT THE INVENTION

The bacterium strain of the present invention (referred to hereinafteras "the present strain") has bacteriological properties as shown below.In this context, strains have been classified with the use of thefollowing culture media 1-19 (numericals are based on % by weight).

Medium 1: meat extract 1.0; bactopeptone 1.0; NaCl 0.5; bactoagar 1.5(pH 7.2)

Medium 2: meat extract 1.0; bactopeptone 1.0; NaCl 0.5 (pH 7.2)

Medium 3: meat extract 1.0; bactopeptone 1.0; NaCl 0.5; gelatin 1.0 (pH7.2)

Medium 4: bactolitmus milk 10.0

Medium 5: bactopeptone 1.0; KNO₃ 0.1

Medium 6: bactopeptone 1.0; NaNO₃ 0.1

Medium 7: bactopeptone 1.0; NaCl 0.5; D-glucose 0.5 (pH 7.0)

Medium 8: bactopeptone 1.0

Medium 9: TSI agar (manufactured by Eiken Chemical Co., Ltd.): indicatedamount

Medium 10: meat extract 1.0; bactopeptone 1.0; NaCl 0.5; soluble starch0.2; bactoagar 1.5 (pH 7.2)

Medium 11: NaNH₄ HPO₄. 4H₂ O 0.15; KH₂ PO₄ 0.1; MgSO₄. 7H₂ O 0.02;citric acid 0.25 (pH 6.8)

Medium 12: Christensen medium (manufactured by Eiken Chemical Co.,Ltd.): indicated amount

Medium 13: D-glucose 1.0; KH₂ PO₄ 0.1; MgSO₄. 7H₂ O 0.05; HCl 0.02;nitrogen sources 0.1 (pH 7.2)

(Nitrogen sources are sodium nitrate and ammonium sulfate.)

Medium 14: Bacto Pseudomonas Ager F medium (manufactured by Difco):indicated amount

Medium 15: urea medium (manufactured by Eiken Chemical Co., Ltd.):indicated amount

Medium 16: paper filter for cytochrome oxidase test (manufactured byNissui Seiyaku)

Medium 17: 3% aqueous hydrogen peroxide

Medium 18: OF basal medium (manufactured by Difco): indicated amount

Medium 19: (NH₄)₂ HPO₄ 0.1; KCl 0.02; yeast extract 0.02; MgSO₄.7H₂ O0.02; bactoagar 2.0; BCP (0.2% solution) 0.4

I. Bacteriological properties

(a) Microscopic observation

Bacillus having a bacterial cell size of 0.6-0.7 82 m×2-3 μm, oneflagellum and mobility. Negative with respect to Gram's stain. Noacidophilicity.

(b) Growth in/on various culture media

(i) Bouillon agar plate culture (medium 1) Good growth. Colonies arecircular with smooth surface and periphery, and they are yellow,translucent and lustrous.

(ii) Bouillon agar slant culture (medium 2) Fair growth. Colonies arefilamentous, lustrous, yellow and translucent.

(iii) Bouillon liquid culture (medium 2) Fair growth with turbidity.

(iv) Bouillon gelatin stab culture (medium 3) Growth on the superficiallayer with gelatin liquefaction.

(v) Litmus milk culture medium (medium 4) Decolored (after seven days)and peptonized.

(c) Physiological properties

(1) Nitrate reduction and denitrification reaction (media 5 and 6)Negative.

(2) MR test (medium 7) Negative.

(3) VP test (medium 7) Negative.

(4) Indole production (medium 8) Negative.

(5) Hydrogen sulfide production (medium 9) Positive.

(6) Starch hydrolysis (medium 10) Positive.

(7) Utilization of citric acid (media 11 and 12) Positive.

(8) Utilization of inorganic nitrogen sources (medium 13) Negative withrespect to nitrates; positive with respect to ammonium salts.

(9) Pigment production (medium 14) Water-insoluble pigment produced.

(10) Urease (medium 15) Negative.

(11) Oxidase (medium 16) Negative.

(12) Catalase (medium 17) Positive.

(13) Range of growth conditions (medium 2)

Growth temperature in the range of from 3° to 49° C., optimally from 23°to 37° C.

Growth pH in the range of from 5 to 10, optimally from 6 to 9.

(14) Behavior toward oxygen Aerobic.

(15) O-F test (medium 18) Oxidized.

(16) Production of acids and gases from saccharides (medium 19)

(+: produced; -: not produced)

    ______________________________________                                                   Production of acid                                                                        Production of gas                                      ______________________________________                                        1.   L-Arabinose +             -                                              2.   D-Xylose    +             -                                              3.   D-Glucose   +             -                                              4.   D-Mannose   +             -                                              5.   D-Fructose  +             -                                              6.   D-Galactose +             -                                              7.   Maltose     +             -                                              8.   Sucrose     +             -                                              9.   Lactose     +             -                                              10.  Trehalose   +             -                                              11.  D-Sorbitol  -             -                                              12.  D-Mannitol  -             -                                              13.  Inositol    -             -                                              14.  Glycerin    +             -                                              15.  Starch      +             -                                              ______________________________________                                    

(17) Ice nucleus activity

The cell solution obtained by the cultivation in medium 2 is dilutedwith 10⁷ times its quantity of distilled water, a 2 ml portion of whichis placed in a test tube having a diameter of 10 mm and cooled at -5° C.for 1 hour. "Positive" indicates "frozen" while "negative" indicates"unfrozen".

Positive. (Control distilled water with no additives is not frozen andthus is negative.)

(18) Phytopathogenicity toward tea Negative.

The test performed in this connection is as follows:

On the leaves of a tea (Thea sinensis L. var. bohea) is inoculated abacterium. The leaves which have been allowed to stand at a temperatureof 27° C. and a humidity of 100% for one day are judged positive if thebacterium is proliferated at the part where it is inoculated whilenegative, if the bacterium is not proliferated.

(19) Tobacco hypersensitivity reaction Negative.

In this context, a test is performed in accordance with the methoddescribed in Phytopathology, 77, 611-615 (1987).

II. Reason for the judgment as a new strain:

(i) Reason for the judgment of the present strain as belonging toXanthomonas campestris:

As a result of the comparison of the above-described bacteriologicalproperties with those described in Bergey's Manual of SystematicBacteriology (1984) and also with those of the known Xanthomonascampestris pv. campestris [Goto Masao et al., Nisshokubyo-Hou, 54, 192(1988)], the present strain was judged as a strain belonging toXanthomonas campestris.

(ii) Reason for the judgment of the present strain as a new strain:

The differences in bacteriological properties between the present strainand the three Xanthomonas campestris strains currently known will beshown below in Table 1.

                  TABLE 1                                                         ______________________________________                                                            Known     Known   Known                                   Items of  Present   strain    strain  strain                                  comparison                                                                              strain    A         B       C                                       ______________________________________                                        Growth rate                                                                             high      normal            low                                     in YP agar                                                                              (25° C. ×                                                                  (28° C. ×                                    plate     1 or 2    3 days)                                                   culture   days)                                                               Growth temp.                                                                  Min.      3° C.                10° C.                           Optimal   23-37° C.            33.5° C.                         Max.      49° C.                                                                           (generally                                                                              (generally                                                                            42-43° C.                                            40° C. at                                                                        40° C. at                                                    maximum)  maximum)                                        Oxidase   negative  negative  positive                                        Utilization                                                                             negative  (generally                                                                              (generally                                      of malonic          utilized) utilized)                                       acid                                                                          ______________________________________                                         Note 1:                                                                       Strain A: obtained from the leaves of Florida citrus [Goto Masao et al.,      NisshokubyoHou, 54, 196 (1988) and Minsavage G.V. and Stall R.E. (1986),      Proc. 6th Int. Conf. Plant Path. Bact.,                                       Strain B: X. campestris pv. translucens [Kim H.K., Orser C., Lindow S.E.      and Sands D.C., Plant Disease, 71, 994-997                                    Strain C: obtained from tea buds (INAX) [Goto Masao et al.,                   NisshokubyoHou, 54, 189-197 (1988)                                             Note 2:                                                                      Growth rate in the YP agar plate culture was judged as follows:               "high": when colonies were formed within 1 or 2 days                          "normal": when they were formed within 3 or 4 days, and                       "low": when they were formed in 5 days or more.                          

As a result of the examination of the difference between the presentstrain and the known strains, the present strain was distinguished fromthe known strains on the points shown in the above table, and thusjudged as a new strain.

The present strain was deposited with Fermentation Research Institute,Agency of Industrial Science and Technology (the name of which waschanged to National Institute of Bioscience and Human-Technology, Agencyof Industrial Science and Technology on Jan. 1, 1993) at 1-3, Higashi 1chome, Tsukuba-shi, Ibaraki-ken, 305 Japan, and accorded the accessionnumber FERM P-12764 on Feb. 17, 1992. In this connection, the presentstrain was transferred on Feb. 17, 1993 to the aforementioned Institutewhich is one of the international deposition authorities according tothe Budapest Treaty, and accorded the accession number FERM BP-4191.

III. Cultivation of the present strain

As a method for the cultivation of the present strain, conventionalmethods for culturing Xanthomonas campestris bacteria can be generallyemployed. A specific example is aerobic cultivation using theaforementioned medium 14 or a liquid medium (medium 20) which is thesame as medium 14 except for agar and conducted at a temperature of 25°C. for 1 or 2 days. In this context, industrially inexpensive sugarssuch as glucose, sucrose, maltose and lactose, although not limited, arepreferably employed as carbon sources. The culture media for the purposeof the present invention may be either liquid media or solid media, andpreferably include, in addition to those described above, a solid mediumsuch as the aforementioned medium 1, a liquid medium such as medium 2which is the same as medium 1 except for agar, and the following medium21:

Medium 21: meat extract 1.0; bacpeptone 1.0; MgSO₄.7H₂ O 0.05; K₂ HPO₄0.05 (pH 7.0)

The present strain produces xanthan gum on the surface of the cells, sothat they adhere easily to each other. On the other hand, as individualcells of the present strain, as will be apparent from the results inExamples 2 and 4, have ice nucleus-forming activity, culturing theindividual cells which have been previously dispersed can be said morepreferable than culturing adhering cells since the former culture willnot require dispersion of the adhered cells and besides can freeze alarge amount of water with a small amount of cells. Further, as will beapparent from the results in Example 3, it is preferred to culture thepresent strain in a medium (liquid medium) containing lactic acid.

In the cultivation of the present strain, the present inventors havealso found, as a result of the following cultivation test, the fact thatculturing the present strain in a medium containing lactic acid canintensively promote the growth of the cells.

Cultivation test

Shake culture as pre-culture was carried out in the aforementionedmedium 21 under aerobic conditions of 25° C. for 48.5 hours (about 2days), and the culture solution thus obtained was inoculated into eachof the liquid media specified in Table 2 below in a proportion of 1.5%before shake culture at 25° C. for 75 hours. Each of the culturesolutions thus obtained was further cultured on an agar plate of medium14 for 72 hours, and the number of cells in each culture was determined.The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                           Number of cells after 72                                   Medium             hours (cells/ml)                                           ______________________________________                                        Basal medium (medium 21)                                                                         1.5 × 10.sup.10                                      Basal medium containing no                                                                       1.5 × 10.sup.10                                      K.sub.2 HPO.sub.4                                                             Basal medium containing no                                                                       1.3 × 10.sup.10                                      MgSO.sub.4.7H.sub.2 O                                                         Basal medium containing 0.1%                                                                     2.0 × 10.sup.10                                      lactic acid                                                                   ______________________________________                                    

It is apparent from the above results that the growth of the presentstrain is intensively promoted by adding lactic acid to the culturemedia. The content of lactic acid in a medium is preferably from 0.02 to0.2%, more preferably from 0.05 to 0.15%.

The present invention also provides an ice nucleus-forming substancecontaining the new ice nucleus-forming bacterium described in the aboveparagraphs I, II and III. In this context, the phrase "containing theice nucleus-forming bacterium" means that the main constituent forforming ice nuclei is Xanthomonas campestris INXC-1, and it is needlessto say that the other components or raw materials may be appropriatelycontained, provided that they do not adversely affect the objects of thepresent invention. Typical examples of the ice nucleus-forming substanceof the present invention include a culture product obtained by culturingthe present strain, living cells collected from the culture product in aconventional manner, and a physical/chemical treatment product of theliving cells as well as a dryed product thereof. The ice nucleus-formingsubstance also includes the so-called dead bacterium, i.e., icenucleus-forming bacterium having no proliferating ability. By treating abacterium under an ultra-high pressure of 300 MPa or more for 1-10minutes, it is possible to kill the microorganism. Such sterilization,although depending on the numbers of cells to be treated, can beperformed, for example, by treating 2.0×10¹⁰ cells/ml under 300 MPa for5 minutes to a survival rate of 1 cell/ml or less. In the ultra-highpressure treatment, the temperature should be set preferably lower than28° C., e.g., in the range of from -20° C. to 28° C., more preferablyfrom 0° C. to 10° C., from the standpoint of the ice nucleus-formingactivity. Alternatively, by adding hydrogen peroxide and then leavingthe resultant mixture to stand for a while, it is also possible to killthe present strain while keeping ice nucleus activity. For example, byadding an aqueous hydrogen peroxide in an amount of from about 0.05 to0.1M as hydrogen peroxide to the culture solution and leaving it tostand at 10° C. for about 4 hours, it is generally possible to killabout 2.0×10¹⁰ cells/ml to a survival rate of 1 cell/ml or less.

The ice nucleus-forming substance of the present invention can formnuclei of ice thereby freezing a substance, and thus it is preferablyused in freezing a liquid food or in manufacturing artificial snow in asnow machine. Although the amount of the ice nucleus-forming substanceused is not particularly limited, it is preferably used in aconcentration such that will not impair the physical properties or tasteof the liquid food in the former case, e.g., about one cell of icenucleus-forming bacterium per ml of the liquid food, or in aconcentration of one cell of ice nucleus-forming bacterium per drop ofthe aqueous suspension (ca. 1×10⁻¹⁰ -1×10⁻⁹ g/drop) in the latter case.

In an actual procedure for using the ice nucleus-forming substance in asnow machine, the ice nucleus-forming substance may be preliminarilyadded to the water to be supplied to the snow machine by which the watercan be sprayed to be changed into snow, and the resultant mixture isthen sprayed. The ice nucleus-forming substance is preferably added in aconcentration such that individual droplets formed by spraying watercontain at least one ice nucleus-forming bacterium. As a means forspraying, a spray nozzle for dual fluids which is suitable for makingdroplets (particle size: generally, 400-5 μm) by mixing air (compressedair) and water can be generally used. The liquid sprayed from the nozzleis diffused into an atmosphere generally cooled to 0° C. or less,preferably to -5° C. or less, and contacted with the cooled atmospherewhereby the water droplets are changed into snow. If the cooledatmosphere cannot be obtained under natural weather conditions, such anatmosphere may be prepared artificially. In preparing such a cooledatmosphere artificially, cooled air prepared, for example, by liquidnitrogen is generally preferably used for spraying where desired.

The ice nucleus-forming substance of the present invention can thus beused for freezing substances and does have the effect of promoting orfacilitating the freezing of substances, and hence can be utilized, inaddition to the cases mentioned above, for a variety of cases in whichthe freezing of substances is involved. Some examples of the utilityforms of the ice nucleus-forming substance of the present inventioninclude: the freezing of substances including foods such as vegetables,fruits, marine products, meats, milk, eggs, cereals, and processed foods(, e.g., ice cream, ice candies, sherbet, juices, sauces, soups and beancurd) by freezing these substances to which the present icenucleus-forming substance has been already added by blending, spreadingor pouring; the concentration of liquid foods such as vegetable juices,fruit juices, milk, fermented milk, egg liquid, alcoholic drinks,coffee, tea extracts, and liquid seasonings (, e.g., cooking vinegar,soy sauce and sauces), or the concentration of liquid substances such asindustrial waste liquid by cooling these substances to which the presentice nucleus-forming substance has been already added, thereby partiallyforming ice and then removing the ice thus formed; the lyophilization ofsubstances including foods such as fruit juices, coffee, tea extracts,liquid or semi-solid seasonings (, e.g., soy sauce, sauces, dressings,broths, fermented soy bean paste, and gelatinizable products such asagar) by lyophilizing by freezing these substances to which the presentice nucleus-forming substance has been already added and vaporizingmoisture from the frozen substances thus obtained; the formation of anartificial ground by freezing the ground to which the present icenucleus-forming substance has been already added; and the preparation ofartificial rain containing the present ice nucleus-forming substance asan ice crystalline nucleus. Another example of the utility formsincludes the softening of green vegetables which comprises spraying thepresent ice nucleus-forming substance over the surfaces of the greenvegetables such as NOZAWANA, and cooling the whole vegetablesartificially or under natural environment thereby to freeze the greenvegetables partially or entirely.

The present invention is further described in detail hereinbelow withreference to Examples and Test Examples.

EXAMPLE 1

(Production of the ice nucleus-forming bacterium of the presentinvention):

New buds of tea obtained in a tea field in Morimachi, Shuchi-gun,Shizuoka-ken, Japan were suspended in a sterilized physiological salinesolution and cultured on a Bacto Pseudomonas Agar F medium (medium 14)at 20° C. for three days to form colonies. These colonies were thensuspended in water for isolating, as positive bacteria, icenucleus-forming bacteria which could freeze upon cooling at -5° C. for 1hour. The results indicated in the paragraph "I. Bacteriologicalproperties" set forth hereinbefore were obtained by examining thebacteriological properties of the thus isolated strain, and the strainwas designated as Xanthomonas campestris INXC-1.

EXAMPLE 2

(i) Preparation of the present ice nucleus-forming substance:

Xanthomonas campestris INXC-1 obtained in Example 1 was inoculated inthe following culture medium 22 and shake-cultured under aerobicconditions at 25° C. for 1 day. By this culture, the concentration ofthe cells in the culture solution reached about 2.0×10¹⁰ cells/ml. Thecells obtained by separation from the culture solution were washed withsterilized distilled water and then diluted with the same distilledwater to obtain a cell solution having the same concentration as theculture solution. The bacterium solution was subjected to pressurizingtreatment at 5° C. under a pressure of 300 MPa for 5 minutes in a highpressure treatment apparatus (MFP-7000) manufactured by Mitsubishi HeavyIndustries, Ltd. to sterilize the cells.

    ______________________________________                                        Medium 22:                                                                    ______________________________________                                        Yeast extract (Difco)                                                                          10     g                                                     Bactopeptone (Difco)                                                                           10                                                           MgSO.sub.4.7H.sub.2 O                                                                          0.5                                                          Lactic acid      1                                                            Water            1000                                                         (pH              7.0)   (The pH was adjusted                                                          with 1N NaOH.)                                        ______________________________________                                    

The sterilized bacterium solution was lyophilized by a conventionalmethod to obtain a powdery ice nucleus-forming substance. In thiscontext, care was taken to maintain the temperature duringlyophilization below 28° C.

(ii) Application examples of the present ice nucleus-forming substance:

Freezing of foods: (a) Freezing or concentration by freezing

To 5 liters of rice vinegar (acidity: 5%) was added 1 ml of thesterilized bacterium solution obtained as above diluted 10³ times, andthe mixture was cooled at -6° C. for one day to form ice. When the icewas removed in a centrifuge for draining water from vegetables, ricevinegar which was concentrated to an acidity of 15% was successfullyobtained in an amount of 1.5 liters.

As another application example, when 1 m of the same bacterium solutionas above diluted 10² times was added to 16 kg of egg white liquid, andthe mixture was cooled to -18° C., the egg white liquid was successfullyfrozen without being supercooled.

On the other hand, rice vinegar to which no ice nucleus-formingsubstance had been added was supercooled at -6° C. and could not freezewithin one day, and egg white liquid was also supercooled at -18° C. andrequired a considerable period of time for its freezing.

As still another application example, the same sterilized bacteriumsolution as was used in the case of rice vinegar was diluted 10³ timesand added to 2.1 liters of white wine (Brix: 8.0, alcohol percentage:13.0%) to a cell concentration of 2.0×10⁴ cells/ml. The mixture wascooled at -10° C. overnight, and ice thus formed was removed by the samecentrifuge as was used in the case of rice vinegar to obtain about 1.2liters of a concentrated wine (Brix: 12.0, alcohol percentage 19.5%).

Alternatively, the lyophilized ice nucleus-forming substance obtained inthe above paragraph (i) can also be used, by suspending it in water at28° C. or lower in advance, for the concentration of cooking vinegar orwine, or for the freezing of egg white liquid in the same manner as isdescribed above.

Freezing of foods: (b) Lyophilization

Two liters of deionized water at 25° C. was poured into a containercontaining 1 kg of tea to soften the leaves by swelling for 40 minutes.Twenty (20) liters of deionized water at 25° C. was then poured onto thetea leaves, and the mixture was stirred for 10 minutes to obtain anextract from the leaves. The extract thus obtained (cold water extract)was centrifuged.

Further, 20 liters of deionized water at 86° C. was poured onto the tealeaves, and the mixture was subjected to extraction for 10 minutes. Thehot water extract was obtained by centrifugation and combined with thecold water extract previously obtained. The entire extract was thencooled to 5° C. A 1 ml portion, diluted 10³ times, of the samesterilized bacterium solution as was used in the above paragraph"Freezing of foods (a)" was added, and the mixture was cooled at -6° C.for 1 day to form ice.

The ice was removed by centrifugation to obtain a 10-fold concentrationof the tea extract, which was then directly lyophilized to prepare aninstant tea.

Production of artificial snow (a):

The sterilized bacterium solution obtained as described in the aboveparagraph (i) was diluted with water, and 50 drops of 5 μl were placedon a glass plate and cooled at -5° C. for 5 minutes.

The number of sterilized bacterial cells per 5 μl and that of frozendrops are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Bacterial cells                                                                              Frozen drops                                                   ______________________________________                                        100            50                                                             10             50                                                             1              50                                                             0.1            9                                                              no addition    0                                                              ______________________________________                                    

These results indicate that none of the drops to which bacterial cellshad not been added was frozen, while the drops containing the icenucleus-forming substance of the present invention were successfullyfrozen.

Therefore, it can be seen that the drops containing the icenucleus-forming substance of the present invention can be used in theproduction of artificial snow by freezing them at a temperature belowthe freezing point but higher than would be required for the dropscontaining no ice nucleus-forming substance.

Alternatively, the lyophilized ice nucleus-forming substance obtained inthe above paragraph (i) can also be used, by suspending it in water at28° C. or lower in advance, for the production of artificial snow in thesame manner as is described above.

Production of artificial snow (b):

A water dilution of the sterilized bacterium solution obtained asdescribed in the above paragraph (i) was sprayed in the field at atemperature of -7° C. using a snow machine equipped with a nozzle fordual fluids and compressed air to make snow artificially.

In this context, the dilution had a cell concentration of about 2.0×10⁶cells/ml. Droplets (waterdrops) produced by the spraying had an averageparticle diameter of about 100 μm.

EXAMPLE 3

(Method for culturing the present ice nucleus-forming bacterium):

The present strain was inoculated separately in the following medium 23to which one of glucose, maltose and sucrose had been added in aconcentration of 0.1% and in the medium 22 to which 0.1% of lactic acidhad been added, and each inoculated medium was shake-cultured at 25° C.for one day.

    ______________________________________                                        Medium 23:                                                                    ______________________________________                                        Yeast extract (Difco)                                                                          10     g                                                     Bactopeptone (Difco)                                                                           10                                                           MgSO.sub.4.7H.sub.2 O                                                                          0.5                                                          Lactic acid      1                                                            Water            1000                                                         (pH              7.0)   (The pH was adjusted                                                          with 1N NaOH.)                                        ______________________________________                                    

Each of the culture solutions was serially diluted 10-fold in testtubes, and the respective dilutions were individually charged in anamount of 2 ml into ten test tubes having a diameter of 10 mm and cooledat -5° C. for 1 hour. For the respective culture solutions, the dilutionmagnification at freezing and the number of frozen tubes at the dilutionmagnification are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Dilution                                                                              Glucose   Maltose   Sucrose Lactic acid                               ______________________________________                                        10.sup.7                                                                              10        10        10      10                                        10.sup.8                                                                              5         4         5       10                                        10.sup.9                                                                              0         0         1       10                                        .sup. 10.sup.10                                                                       0         0         0       10                                        ______________________________________                                    

It is apparent from th results shown in Table 4 that the higher thedilution magnification, the lower the ice nucleus-forming activity inthe case of the addition of sugar in comparison with the case of theaddition of lactic acid. Just after the cultivation, it was observed inthe case of the addition of glucose, maltose or sucrose that the cellsbegan to agglomerate and soon precipitated when left standing. However,in the case of the addition of lactic acid, no agglomeration occurredand thus no precipitation was observed. Therefore, it has been foundthat, when cultivation is conducted in the presence of lactic acid, thepresent strain does not agglomerate but exhibits ice nucleus-formingactivity even at a higher dilution magnification.

EXAMPLE 4

Xanthomonas campestris INXC-1 obtained in Example 1 was inoculated inmedium 22 and cultured under aerobic conditions at 25° C., for 1 day.After the cultivation (cell concentration: 2.0×10¹⁰ cells/ml), the cellswere washed with sterilized distilled water and diluted with the samedistilled water to obtain a cell solution having the same concentrationas the culture solution. The bacterium solution was pressurized underthe conditions of 100-400 MPa at 5° C. for 5 minutes in a high pressuretreatment apparatus (MFP-7000; manufactured by Mitsubishi HeavyIndustries, Ltd.). The number of viable cells after each treatment wasdetermined by an agar plate culture method. The results are shown inTable 5.

                  TABLE 5                                                         ______________________________________                                        Pressurizing conditions                                                                       Viable cells (per ml)                                         ______________________________________                                        non-treated     2.0 × 10.sup.10                                         100 MPa         1.5 × 10.sup.10                                         200 MPa         1.2 × 10.sup.3                                          300 MPa         <1                                                            400 MPa         <1                                                            ______________________________________                                    

It is apparent from the results of Table 5 that pressurizing treatmentunder 300 MPa for 5 minutes kills the present strain to 1 cell/ml orless. In this context, even in this case, in respect of icenucleus-forming activity, there is no difference between the sterilizedstrain and the present living strain.

Industrial Applicability

The new strain Xanthomonas campestris INXC-1 of the present invention isnot only negative with respect to phytopathogenicity toward tea, butalso negative with respect to tobacco hypersensitivity reaction in whichbacteria which are negative with respect to tobacco hypersensitivityreaction are generally free of phytopathogenicity. Besides, this newstrain is an ice nucleus-forming bacterium having a high growth rateduring culture, so that it has become possible for the first time toprovide a novel ice nucleus-forming bacterium and ice nucleus-formingsubstance by which the objects of the present invention have beenaccomplished. Therefore, the ice nucleus-forming substance of thepresent invention can be expected to be applicable much more extensivelyto a variety of fields in which the freezing of substances is involvedas compared with conventional ice nucleus-forming bacteria.

What is claimed is:
 1. A biologically pure bacterium strain Xanthomonascampestris FERM BP-4191.
 2. A method for culturing bacterium strainXanthomonas campestris FERM BP-4191 which comprises culturing saidstrain in a medium comprising 0.02 to 0.2% by weight of lactic acid. 3.A method for culturing bacterium strain Xanthomonas campestris FERMBP-4191 which comprises culturing said strain in a medium comprising0.05 to 0.15% by weight of lactic acid.
 4. A method for cultivatingbacterium strain Xanthomonas campestris FERM BP-4191 comprisingculturing said strain in a medium comprising 0.1% -0.2% by weight oflactic acid.